Fanconi anemia links reactive oxygen species to insulin resistance and obesity

Abstract

Aims: Insulin resistance is a hallmark of obesity and type 2 diabetes. Reactive oxygen species (ROS) have been proposed to play a causal role in insulin resistance. However, evidence linking ROS to insulin resistance in disease settings has been scant. Since both oxidative stress and diabetes have been observed in patients with the Fanconi anemia (FA), we sought to investigate the link between ROS and insulin resistance in this unique disease model.

Results: Mice deficient for the Fanconi anemia complementation group A (Fanca) or Fanconi anemia complementation group C (Fancc) gene seem to be diabetes-prone, as manifested by significant hyperglycemia and hyperinsulinemia, and rapid weight gain when fed with a high-fat diet. These phenotypic features of insulin resistance are characterized by two critical events in insulin signaling: a reduction in tyrosine phosphorylation of the insulin receptor (IR) and an increase in inhibitory serine phosphorylation of the IR substrate-1 in the liver, muscle, and fat tissues from the insulin-challenged FA mice. High levels of ROS, spontaneously accumulated or generated by tumor necrosis factor alpha in these insulin-sensitive tissues of FA mice, were shown to underlie the FA insulin resistance. Treatment of FA mice with the natural anti-oxidant Quercetin restores IR signaling and ameliorates the diabetes- and obesity-prone phenotypes. Finally, pairwise screen identifies protein-tyrosine phosphatase (PTP)-α and stress kinase double-stranded RNA-dependent protein kinase (PKR) that mediate the ROS effect on FA insulin resistance.

Innovation: These findings establish a pathogenic and mechanistic link between ROS and insulin resistance in a unique human disease setting.

Conclusion: ROS accumulation contributes to the insulin resistance in FA deficiency by targeting both PTP-α and PKR.

FIG. 1.

RTK array identifies defective IR signaling in FA cells. (A–D) Human normal lymphoblastic cell line HSC93 (A, B) and FA-C patient-derived lymphoblastic cell line HSC536 (C, D) were treated with (A, C) or without (B, D) H₂O₂ (0.5 mM) for 15 min, and WCEs were subject to RTK array analysis. Phosphorylation status was determined by subsequent incubation with biotin-conjugated anti-phosphotyrosine antibody and streptavidin-linked horseradish peroxidase. Each RTK is spotted in duplicate, and the positive control includes six dots of the first layer and two dots of the last layer. (E) The indicated cells were treated with or without H₂O₂ (0.5 mM) or insulin (10 μg/ml) for 15 min. WCEs were separated by SDS-PAGE and probed with antibodies against phospho-IR and IR, and actin as loading control. FA, Fanconi anemia; H₂O₂, hydrogen peroxide; IR, insulin receptor; RTK, receptor tyrosine kinase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; WCEs, whole-cell extracts.

FIG. 2.

FA mice seem to be diabetes mellitus prone. (A) Whole-blood glucose was measured in mice fasted overnight by using tail blood (n=6–8 per genotype). (B, C) After having been fasted, mice (4–5 months old) were given an injection (IP) of either glucose (1 g/Kg of body weight) (B) or insulin (1.0 U/Kg of body weight) (C), and whole-blood glucose was examined at the indicated time points by using the Glucometer. The results are shown as the percentage of the initial glucose levels. *p<0.05, ***and p<0.001for Fanca^−/− or Fancc^−/− mice versus wt mice. (D) Increased acute-phase insulin release in FA mice. Mice were IP injected with glucose (1 g/kg body weight), and whole blood was collected from tail vein samples at the indicated times for insulin measurements. (E) Mice were fasted overnight, and whole blood was collected from tail vein samples at the indicated times for insulin measurements. Values are means±SD (n=4–6). *p<0.05 versus WT. FANCA, Fanconi anemia complementation group A; FANCC, Fanconi anemia complementation group C; IP, intraperitoneal; WT, wild-type.

FIG. 3.

FA mice show a high risk for diabetes induced by a high fat diet. (A) Images of embryos of the indicated genotypes at E12.5. Genotypes of the embryos were determined by PCR. (B–D) Size comparison between wt (+/+) and FA (−/−) mice at day one (B) or day 30 (C) after birth, or 4 months after being fed with HFD (D). (E) wt or FA mice were fed with HFD starting at 7 weeks old, and the weight was examined at the indicated time points. The results are shown as the original readout (left) and fold increase from the starting weight (right). *p<0.05 for Fanca^−/− or Fancc^−/− mice versus wt mice. (F) Mice (8–9 weeks old, n=6–8 per genotype) were fed with HFD for 2 months, fasted overnight, and whole-blood glucose was measured using tail blood. *p<0.05; **p<0.01 for a comparison between groups of mice fed with HFD for 9 weeks versus before the feeding. (G) HFD-fed mice described in (F) were fasted overnight and given an injection (i.p) of insulin (1.0 U/Kg), and whole-blood glucose was examined at the indicated time points by using the glucometer. The results are shown as the original readout. *p<0.05 for Fanca^−/− or Fancc^−/− mice versus wt mice. HFD, high-fat diet. (To see this illustration in color the reader is referred to the web version of this article at www.liebertpub.com/ars).

FIG. 4.

Decreased IR^{Tyr1158/1162/1163} phosphorylation in FA mice. (A, B) Fanca^−/− (A) or Fancc^−/− (B) and wt littermates were fasted overnight followed with an injection of saline or insulin (5 U/Kg of body weight), and tissue samples were collected 10 min after the injection. Western blot analysis of IR and AKT was performed by separating freshly prepared tissue homogenates on an SDS-PAGE gel and probed with antibodies against phosphorylated IR (p-IR) Tyr1158/1162/1163 and total IR (beta-subunit), or AKTSer308 (p-AKT) and total AKT. The data are presented as mean±SD. *p<0.05, **p<0.01 for Fanca^−/− or Fancc^−/− mice versus WT mice. (C) HepG2 cells transduced with lentivirus carrying shRNA for the human FANCA or FANCC gene were starved overnight and treated with insulin (10 μg/ml) for 15 min. Cell lysates were separated with SDS-PAGE and probed with antibodies against p-IR, IR, p-AKT, AKT, FANCA, FANCC, and actin. Protein band intensities were analyzed using ImageJ software. The level of each phosphorylated form or its corresponding total protein was first normalized to that of actin and then, the ratio for the phosphorylated form/total protein was calculated. The data are presented as mean±SD. *p<0.05, **p<0.01 versus the control groups.

FIG. 4.

Decreased IR^{Tyr1158/1162/1163} phosphorylation in FA mice. (A, B) Fanca^−/− (A) or Fancc^−/− (B) and wt littermates were fasted overnight followed with an injection of saline or insulin (5 U/Kg of body weight), and tissue samples were collected 10 min after the injection. Western blot analysis of IR and AKT was performed by separating freshly prepared tissue homogenates on an SDS-PAGE gel and probed with antibodies against phosphorylated IR (p-IR) Tyr1158/1162/1163 and total IR (beta-subunit), or AKTSer308 (p-AKT) and total AKT. The data are presented as mean±SD. *p<0.05, **p<0.01 for Fanca^−/− or Fancc^−/− mice versus WT mice. (C) HepG2 cells transduced with lentivirus carrying shRNA for the human FANCA or FANCC gene were starved overnight and treated with insulin (10 μg/ml) for 15 min. Cell lysates were separated with SDS-PAGE and probed with antibodies against p-IR, IR, p-AKT, AKT, FANCA, FANCC, and actin. Protein band intensities were analyzed using ImageJ software. The level of each phosphorylated form or its corresponding total protein was first normalized to that of actin and then, the ratio for the phosphorylated form/total protein was calculated. The data are presented as mean±SD. *p<0.05, **p<0.01 versus the control groups.

FIG. 5.

TNF-α-generated ROS contribute to decreased-IR signaling. (A) Beta cells isolated from wt and Fanca^−/− mice were treated with saline, TNF-α, Quercetin, or TNF-α and Quercetin. Cells were labeled with FITC-conjugated CM-H2DCFDA, and ROS production was examined by flow cytometry. (B) Beta cells were treated with or without TNF-α for 30 min, followed by insulin (10 μg/ml) for 15 min. Quercetin treatment was given 30 min before TNF-α treatment. Cell lysates were analyzed by immunoblotting. (C) The indicated mice (2–3 months old, n=6–8 each group) were injected with Quercetin (50 mg/Kg) twice per week for 3 weeks, followed by a single TNF-α (100 μg/Kg) injection. Liver, fat, and muscle were collected, and a single-cell suspension was prepared. ROS production was examined by flow cytometry with H2DCFDA staining. (D) Mice were treated as described in (C), fasted overnight after the final injection, and given an injection of insulin (5 U/Kg). After 10 min, the liver, fat, and muscle were collected, and the expression of insulin signaling components was analyzed by immunoprecipitation and western blotting. Protein band intensities were analyzed using ImageJ software. The level of each phosphorylated form or its corresponding total protein was first normalized to that of actin and then, the ratio for the phosphorylated form/total protein was calculated. The data are presented as mean±SD. *p<0.05, **p<0.01 for Fanca^−/− mice versus WT littermates. FITC, fluorescein isothiocyanate; ROS, reactive oxygen species; TNF-α, tumor necrosis factor alpha.

FIG. 5.

TNF-α-generated ROS contribute to decreased-IR signaling. (A) Beta cells isolated from wt and Fanca^−/− mice were treated with saline, TNF-α, Quercetin, or TNF-α and Quercetin. Cells were labeled with FITC-conjugated CM-H2DCFDA, and ROS production was examined by flow cytometry. (B) Beta cells were treated with or without TNF-α for 30 min, followed by insulin (10 μg/ml) for 15 min. Quercetin treatment was given 30 min before TNF-α treatment. Cell lysates were analyzed by immunoblotting. (C) The indicated mice (2–3 months old, n=6–8 each group) were injected with Quercetin (50 mg/Kg) twice per week for 3 weeks, followed by a single TNF-α (100 μg/Kg) injection. Liver, fat, and muscle were collected, and a single-cell suspension was prepared. ROS production was examined by flow cytometry with H2DCFDA staining. (D) Mice were treated as described in (C), fasted overnight after the final injection, and given an injection of insulin (5 U/Kg). After 10 min, the liver, fat, and muscle were collected, and the expression of insulin signaling components was analyzed by immunoprecipitation and western blotting. Protein band intensities were analyzed using ImageJ software. The level of each phosphorylated form or its corresponding total protein was first normalized to that of actin and then, the ratio for the phosphorylated form/total protein was calculated. The data are presented as mean±SD. *p<0.05, **p<0.01 for Fanca^−/− mice versus WT littermates. FITC, fluorescein isothiocyanate; ROS, reactive oxygen species; TNF-α, tumor necrosis factor alpha.

FIG. 6.

Quercetin attenuates the diabetes phenotype in FA mice. (A) Fanca+/+ and Fanca^−/− mice (3–4 months old, n=6–8 each group) were injected with Quercetin (50 mg/Kg) twice per week for 2 weeks. After the final injection, whole-blood glucose was measured in mice fasted overnight by using tail blood. The data are presented as mean±SD. *p<0.05 for Fanca^−/− mice versus Fanca+/+mice. (B, C) The mice as described in (A) were fasted and given an injection (IP) of either glucose (1 g/Kg of body weight) (B) or insulin (1.0 U/Kg of body weight) (C), and whole-blood glucose was examined at the indicated time points by using the glucometer. The results are shown as the percentage of the initial glucose levels. *p<0.05 for Fanca^−/− mice versus Fanca+/+mice, or Fanca^−/− mice injected with Quercetin versus those with vehicle. (D) Fanca+/+and Fanca^−/− mice (6–7 weeks old, n=6–8 each group) were fed with HFD and injected with Quercetin (50 mg/Kg) twice per week for 6 weeks. The weight was examined at the indicated time points of the Quercetin treatment. The results are shown as the original readout. *p<0.05 for Fanca^−/− fed with HFD without Quercetin treatment versus Fanca+/+mice.

FIG. 7.

Pair-wise screen identifies PTP-α and PKR inhibitors that rescue insulin resistance. (A) HepG2 cells transduced with a lentivirus carrying shRNA for the FANCA gene were pretreated with or without the PKR inhibitor (5 μM) for 30 min, followed by TNF-α at 10 μg/ml for 30 min, and insulin (10 μg/ml) for another 15 min. Cell lysates were analyzed with antibodies against p-IR, IR, p-IRS-1, IRS-1, PKR, or Actin. (B) The cells described in (A) were transduced with a lentivirus carrying shRNA for PTP-α and treated with or without insulin (10 μg/ml) for 15 min. Immunoprecipitation and immunoblotting were used to analyze p-IR and p-IRS-1 as well as PTP-α and Actin. (C) The cells described in (B) were pretreated with the PKR inhibitor II (5 μM) for 30 min, followed by TNF-α (10 μg/ml) for 30 min, and insulin (10 μg/ml) for another 15 min. Cell lysates were separated by SDS-PAGE and probed with antibodies against the indicated antibodies. Protein band intensities were analyzed using ImageJ software. The level of each phosphorylated form or its corresponding total protein was first normalized to that of actin and then, the ratio for the phosphorylated form/total protein was calculated. The data are presented as mean±SD. *p<0.05, **p<0.01 versus the control groups. (D) A model for ROS-induced insulin resistance in FA. In this model, the overproduction of TNF-α results in the accumulation of ROS, a physiological phenomenon often found in FA patients. Elevated accumulation of ROS would affect two critical steps of IR signaling: (i) decrease IR tyrosine phosphorylation through PTP-α; (ii) increase inhibitory phosphorylation of IRS-1 by activation of PKR kinase, leading to insulin resistance and obesity. The natural anti-oxidant Quercetin proves effective in antagonizing the negative effect of ROS on insulin signaling and maintaining metabolic homeostasis. IRS, insulin receptor substrate; PKR, double-stranded RNA-dependent protein kinase; PTPs, protein-tyrosine phosphatases.